Prevalence rate of laboratory defined aspirin resistance in cardiovascular disease patients: A systematic review and meta-analysis

Background: Cardiovascular disease (CVD) is the first cause of mortality worldwide, with all the healthcare systems facing this very challenging issue. Aspirin continues to be the major gold-standard treatment worldwide in the prevention of thrombotic disease in patients with CVD, even though not all individuals respond to antiplatelet therapy in a similar way, being resistant to aspirin. The aim of this study was to determine the prevalence of laboratory defined aspirin resistance in CVD patients worldwide. Methods: Relevant articles were identified through searching EMBASE, PubMed/ MEDLINE, ISI /Web of Science, Scopus, and the Cochrane Library, from January 2000 to February 2018. The methodological quality of the included studies was critically appraised using the Newcastle-Ottawa scale. The pooled prevalence of laboratory defined aspirin resistance was computed using the Der Simonian-Laird random-effect model. Results: We included 65 studies, with a total of 10,729 patients. The overall prevalence of laboratory defined aspirin resistance in CVD patients was 24.7% ([95%CI 21.4-28.4]. Women were found to be at increased risk of laboratory defined aspirin resistance compared to men, with an odds ratio of 1.16 [95%CI 0.87-1.54] Conclusion: Doctors and healthcare providers should pay special attention to aspirin resistance since lack of awareness could cause problems and increase mortality in these patients, if not properly treated with higher aspirin doses.

From a biochemical standpoint, aspirin inhibits the conversion of arachidonic acid to thromboxane A2, the main metabolite of prostaglandin synthesis, via cyclooxygenase (COX) (5). Even low daily aspirin doses (in the range 75-150 mg) are able to suppress biosynthesis of thromboxane, inhibiting the accumulation of platelets, and reducing the risk of CVD (6). However, aspirin does not always prevent the formation of thromboxane A2 due to failure to inhibit platelet COX (7). As such, all individuals do not respond to antiplatelet therapy in a similar way; some people suffer from thromboembolic events despite ongoing antiplatelet therapy (8,9). The mechanism of resistance to aspirin is still unclear. Different patients may require different doses of aspirin to inhibit platelet function (10) and this calls up for a personalized treatment.
Several studies have been conducted to evaluate the rate of resistance to aspirin in CVD patients. Therefore, the aim of this study was to determine the prevalence of aspirin resistance by conducting a systematic review and metaanalysis of aspirin resistance in CVD patients worldwide.

Methods
The research question of the present work is the worldwide prevalence rate of laboratory defined aspirin resistance in CVD patients. This is a systematic review and meta-analysis that identified aspirin resistance studies with an assessment of its adverse effects on cardiovascular patients. There are several measurement methods to investigate platelet function test, Findings showed that the blood test is more sensitive than urine level, therefore, the present study mostly used two methods of platelet function test and verify now aspirin assay, Some studies also used platelet aggregation multiple method (11). Findings of this study were reported on the basis of the "Preferred Reporting Items for Systematic Reviews and Meta-Analyses"(PRISMA) guidelines (12). We searched different scholarly electronic databases, such as EMBASE, PUBMED/MEDLINE, ISI/Web of Science, Scopus, and the Cochrane Library, from January 2000 to February 2018. To find more potentially relevant studies, the reference list of the included studies was also hand-searched. After the search, all records were entered to the EndNote Reference Manager X8. At this point, all duplicate articles were deleted. Using the Boolean operators (AND, OR), the search strategy was performed as follows: ("platelet resistance" OR "drug resistance" OR "acetylsalicylic acid" OR aspirin OR "antiplatelet platelets" OR "aspirin resistance") AND ("cardiovascular disease" OR "ischemic heart disease" OR "acute coronary syndrome"). A total of 2047 studies were reached from databases search, after deletion of the number 650 duplicates, 987 unrelated studies and 204 articles on the base abstract were excluded, 138 studies were included to title and abstract screening. In addition, we found 32 studies based on other sources. A total of 138 studies full texts were resumed and reviewed based on inclusion criteria. Finally, 65 studies with 10,729 participants were subjected.
Studies were included if: i) designed as cross-sectional, cohort or case-control investigations; ii) studies whose data were appropriate for the calculation of the prevalence rate; iii) patients with a proper clinically established diagnosis of CVD; and iv) peer reviewed studies published in English. Studies were excluded if: i) designed as letters to editor, editorials, commentaries, case reports or case series and reviews; ii) overlapping studies (in case of repeated/ duplicate/redundant studies, the most comprehensive ones were selected); iii) studies whose data did not allow the calculation of the prevalence rate; and iv) studies whose fulltext could not be accessed Two of the authors independently selected the studies on the basis of these criteria, and in case of disagreement, a third person was used as the referee and eventually resolved the issue through discussion. Data extraction: After selecting the studies, two authors independently extracted and collected the data from the included studies: namely, the surname of the first author of the article, year of publication, country of study, number of participants in the study (based on gender, if available), type of laboratory-defined aspirin resistance, prevalence rate, and mean age or age range of participants. Before analysis we certified the precision of the data. We revised any unequal data and adjusted accordingly. Assessment of methodological quality: The methodological quality of the included studies was critically appraised using the Newcastle-Ottawa (NOS) scale (13). Three, two and five stars were assigned to the scale items based on the three domains (selection of study participants, control of confounders and outcome of interest), respectively. Based on the overall score, studies were divided into three groups: high (1-4 stars), medium (5-7 stars) and low (8-10 stars) bias.

Statistical analysis:
The pooled prevalence of laboratorydefined aspirin resistance was computed using the Der Simonian-Laird random-effect model with its 95% confidence interval (CI) (14). To calculate the effect size (ES), the total sample size and the number of laboratorydefined aspirin resistance patients were used. The I 2 test was used to evaluate heterogeneity between studies, which was classified as low, moderate and high (25%, 50% and 75%, respectively) (15). To assess, the role of variables such as sample size, or geographic area of studies was conducted. To ensure the stability of the results and to investigate the impact of each study on the final outcome, a sensitivity analysis was performed. To examine the effect of gender in laboratory-defined aspirin resistance, odds ratio (OR) was calculated. Also, studies were ranked based on the year of publication and cumulative meta-analysis was conducted to examine the trend of changes over time. Visual inspection of the funnel plot and Egger's regression test were used to evaluate the publication bias (16). Figures with p<0.05 were considered statistically significant. All statistical analyses were conducted with the commercial software comprehensive meta-analysis (CMA) Version 2.

Results
After the initial search of the databases, out of a list of 2079 items, 65 studies were included and analyzed based on the above-mentioned inclusion/exclusion criteria (figure 1) . The overall number of CVD patients was 10,729. Appendix 1 shows the characteristics of the studies retained in the current systematic review and meta-analysis.  Figure 2).   3). This finding showed that women are at increased risk of laboratory-defined aspirin resistance compared to men. Results of cumulative meta-analysis for the prevalence of in patients with cardiovascular disease: The studies were ranked according to the year of publication and cumulative meta-analysis was performed. The results did not change before and after this analysis, and the prevalence was 24.7% ]. Appendix 2 shows cumulative metaanalysis based on the year of publication. Studies were also ranked by sample size. The results did not change before and after the cumulative meta-analysis and the prevalence was stable. Appendix 3 shows cumulative meta-analysis based on the year of publication.
Results of sensitivity analysis for the prevalence of aspirin resistance in patients with cardiovascular disease: Sensitivity analysis was carried out to ensure the stability of the results of the studies. The prevalence of aspirin resistance before and after the sensitivity analysis did not change with the exclusion of each study (Appendix 4). Publication bias: The Egger's regression test results are presented in Appendix 5. Observation of the asymmetry of the funnel plot indicated that there was an evidence of publication bias (p=0.38).

Discussion
The aim of this study was to determine the prevalence rate of laboratory defined aspirin resistance in CVD patients worldwide. The concept laboratory defined aspirin resistance has been argued since 1980s, but discussions in late literature have centralized on evidence why aspirin resistance is probably a mistake (82,83). To the best of our knowledge, systematic search of the literature, meta-analysis and extensive statistical analyses (sub-group analysis, sensitivity analysis, cumulative meta-analysis) were the major strengths of this study. The findings showed that the prevalence of laboratory-defined aspirin resistance in CVD patients was 24.7%, with a higher rate among women. This study, pooling together different investigations reporting conflicting results, has enabled to overcome their statistical limitations and shortcomings. Some studies have, indeed, found that women have more or equal responsiveness rate to aspirin than men, being successful in controlling the COX-1 pathway, whilst other studies have shown no difference between female and male (7). According to other scholars, women would have a worse prognosis than men, whereas other studies reported that the biochemical mechanism of laboratory-defined aspirin resistance is unknown, even though female sex hormones may play an important role (84,85). We computed an OR of 1.16 [95%CI 0.87-1.54], showing that women are at increased risk of laboratorydefined aspirin resistance compared to men.
Another important finding of the study is that, the prevalence rate is different in different regions of the world, putatively because of differences in the biological and genetic make-up of individuals. A higher prevalence was found in Asia, while the lowest rate was computed for studies carried out in America.
These findings pave the way for a personalized treatment, in that individual factors seem to affect the response to aspirin. Clinically speaking, there are some conditions known for predisposing patients to higher rate of aspirin resistance. For instance, several studies have shown that patients undergoing coronary artery bypass grafting (CABG), which results in endothelial tissue damage to the saphenous vein graft, or coronary interventions, are more likely to become resistant to aspirin, with high thrombin level and platelet activation (10,86). This suggests that, after CABG surgery or other interventions, patients should be closely monitored and should receive plavix, alternatively, anti-thrombotic drugs. Usually, aspirin resistance after a CABG surgery persists for a short term period (22,87). This temporal laboratory-defined aspirin resistance was in a population of patients who had withstand coronary bypass. Although no adaptation with treatment is a momentous cause of laboratory aspirin resistance, patient dependency treatment was determined in few studies (88)(89)(90). Cotter et al, have indicated no adaptation to treatment is a significant moderator of negligible consequence. It is substantial to appraise whether patients take their medicines in clinical conditions or in studies that measure the effect of prescription drugs (89,90). According to research, another strategy to control laboratory defined aspirin resistance is the administration of vitamin D (91). Furthermore, patients not practicing enough physical activity and/or with increased blood glucose should require higher aspirin doses (92,93).
However, despite its strengths, the present systematic review and meta-analysis suffers from some limitations, that hinder generalization of the present findings and call up for caution in interpreting results. The major drawback is given by the heterogeneity between studies and the evidence of publication bias. Another limitation is given by the methodological and quality differences among the studies. As such, further larger high-qualities studies in the field are warranted. Moreover, available study data did not allow to investigate the impact of possible risk factors associated with the prevalence of laboratory-defined aspirin resistance.
The findings of the present systematic review and metaanalysis showed that the prevalence of laboratory defined aspirin resistance in CVD patients was 24.7%. Doctors and healthcare providers should pay special attention to this, since lack of awareness could cause problems and increase mortality in these patients, if not properly treated with higher aspirin doses. It is suggested that one way to overcome the problem of laboratory defined aspirin resistance perhaps is to give the patient more medicine. However, this cannot be the result of the study, and more specific studies are required, in the way that the method of platelet related assay, the length of treatment and the amount of drug in patient have the same conditions.